Meiosis is the specialized cellular program that produces haploid male and female gametes from a diploid parental cell as required for sexual reproduction. Our research addresses the central unique feature of this program, a complex series of interactions between maternal and paternal homologs, in three areas: (I) Recombination-independent pairing. This process, which also occurs in mitotically-dividing cells of some organisms ("somatic pairing"), is one of the major chromosomal behaviors for which there is no hint of the fundamental molecular mechanism. We will apply high-throughput 3C analysis to identify pairing "hot spots" and to examine the roles of specific variables of interest to pairing at a single defined locus. We will further analyze pairing interactions by motion correlation analysis of GFP-tagged loci in living cells. As time permits, we will begin to investigate the possibility of isolating (and then characterizing) paired molecules. (II) Recombination-related events. We will continue to analyze the basic nature of recombination at the DNA level by isolation and AFM visualization of recombination intermediates (notably double Holliday junctions) from a single recombination "hot spot". We will initiate a study of crossover interference by searching for functions which, when overproduced, increase or decrease the robustness of this process; also, we will try to distinguish among possible models for interference by creating, and assessing the effects of, a defined "loop" along a chromosome. Finally, we will continue to explore the roles of axis components for recombination by: studies of the "mitotic" cohesin Mcd1/Scc1/Rad21; high resolution analysis of local variations in protein localization via linked MNase studies; and further definition of chromosome organization via high resolution analysis of cohesin and condensin localization along pachytene chromosomes. (III) Motion and mechanics. We will improve our methodology for isolation of individual pachytene chromosomes as objects for mechanical studies. We will pursue our recent discovery of actin- and telomere-based chromosomal motion using existing reagents and by searching for "motion mutants". Defects in meiotic interhomolog interactions underlie many types of hereditary infertility as well as most chromosomal aneuploidies, all of which confer significant societal burdens (e.g. Downs syndrome). Basic understanding of these processes is crucial for diagnosis and potential amelioration of these conditions. [unreadable] [unreadable] [unreadable]